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A reference human induced pluripotent stem cell line for large-scale collaborative studies.

ABSTRACT: Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

SUBMITTER: Pantazis CB

PROVIDER: S-EPMC9782786 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Pantazis Caroline B CB Yang Andrian A Lara Erika E McDonough Justin A JA Blauwendraat Cornelis C Peng Lirong L Oguro Hideyuki H Kanaujiya Jitendra J Zou Jizhong J Sebesta David D Pratt Gretchen G Cross Erin E Blockwick Jeffrey J Buxton Philip P Kinner-Bibeau Lauren L Medura Constance C Tompkins Christopher C Hughes Stephen S Santiana Marianita M Faghri Faraz F Nalls Mike A MA Vitale Daniel D Ballard Shannon S Qi Yue A YA Ramos Daniel M DM Anderson Kailyn M KM Stadler Julia J Narayan Priyanka P Papademetriou Jason J Reilly Luke L Nelson Matthew P MP Aggarwal Sanya S Rosen Leah U LU Kirwan Peter P Pisupati Venkat V Coon Steven L SL Scholz Sonja W SW Priebe Theresa T Öttl Miriam M Dong Jian J Meijer Marieke M Janssen Lara J M LJM Lourenco Vanessa S VS van der Kant Rik R Crusius Dennis D Paquet Dominik D Raulin Ana-Caroline AC Bu Guojun G Held Aaron A Wainger Brian J BJ Gabriele Rebecca M C RMC Casey Jackie M JM Wray Selina S Abu-Bonsrah Dad D Parish Clare L CL Beccari Melinda S MS Cleveland Don W DW Li Emmy E Rose Indigo V L IVL Kampmann Martin M Calatayud Aristoy Carles C Verstreken Patrik P Heinrich Laurin L Chen Max Y MY Schüle Birgitt B Dou Dan D Holzbaur Erika L F ELF Zanellati Maria Clara MC Basundra Richa R Deshmukh Mohanish M Cohen Sarah S Khanna Richa R Raman Malavika M Nevin Zachary S ZS Matia Madeline M Van Lent Jonas J Timmerman Vincent V Conklin Bruce R BR Johnson Chase Katherine K Zhang Ke K Funes Salome S Bosco Daryl A DA Erlebach Lena L Welzer Marc M Kronenberg-Versteeg Deborah D Lyu Guochang G Arenas Ernest E Coccia Elena E Sarrafha Lily L Ahfeldt Tim T Marioni John C JC Skarnes William C WC Cookson Mark R MR Ward Michael E ME Merkle Florian T FT

Cell stem cell 20221201 12

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including ...[more]

PMID: 36459969

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Project description:BackgroundConsiderable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons.MethodsThe NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively.ResultsThe optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons.ConclusionsWe developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of high-quality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future.

Dataset Information

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Publications

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

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